The mid- to late- 1980's were witness to a dramatic increase in the incidence of rheumatic fever and other severe and invasive diseases caused by the gram positive bacterium Streptococcus pyogenes. The reason for this increase is unknown, which to a large extent, reflects our poor understanding of the molecular events that occur between S. pyogenes and host cells during infection and a lack of sophisticated techniques for the molecular genetic analysis of this organism. To begin to address these problems, I am interested in characterizing the initial encounter between a host cell and S. pyogenes in molecular detail and in developing techniques to facilitate this analysis. I have recently identified protein F, a streptococcal fibronectin-binding protein that is an adhesin for certain epithelial cell populations. Expression of the gene which encodes protein F (prtF) is regulated via two independent pathways: One involves oxygen via a signaling mechanism that senses superoxide (O2-) while the second utilizes the regulatory gene rofA. The latter pathway was discovered in a strain which expresses prtF constitutively (i.e. in the absence of an 02- signal), apparently as a result of aberrant expression of rofA. Further characterization of the regulation of prtF represents an important opportunity to gain considerable insight into the bacterial, environmental and host factors which contribute to the dynamics of streptococcal/host cell interaction. It is also a goal of this proposal to develop several new techniques for the identification of virulence and regulatory genes in S. pyogenes. Novel derivatives of Tn916 and Tn4001 will be constructed and used to determine if the constitutive expression phenotype is the result of a mutation in rofA itself, or resident in another gene. Also, since mutants I have constructed that are aberrant in expression of superoxide dismutase (Sod) are also aberrant in expression of prtF, I will develop genetic strategies to test the hypothesis that an O2- sensing global regulatory system plays a key role in streptococcal/host cell interaction through the identification of genes which regulate expression of prtF and sod. Since O2- and rofA do regulate prtF, and expression of protein F also requires atypical culture conditions, identification of other rofA and O2- regulated genes will likely result in the identification of new potential virulence factors. Finally, since rofA likely plays a role in signal transduction, several potential environmental signals for rofA will be evaluated in order to gain insight into rofA 's contribution to pathogenesis.